Vaccine delivery systems using yeast cell wall particles

ABSTRACT

The present invention generally relates to compositions and methods for delivering a vaccine. The compositions and methods disclosed herein are particularly useful in making prophylactic and therapeutic vaccines.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. application Ser. No.16/227,468, filed Dec. 20, 2018, which is a Divisional of U.S.application Ser. No. 15/123,479, which is the U.S. National Stageapplication of PCT/US2015/018728, filed Mar. 4, 2015, which claimspriority from U.S. Provisional Application Nos. 61/948,504, filed Mar.5, 2014, and 62/060,124, filed Oct. 6, 2014.

FIELD OF THE INVENTION

The present invention generally relates to compositions comprising yeastcell wall particles and methods for delivering a vaccine. Thecompositions and methods disclosed herein are particularly useful inmaking prophylactic and therapeutic vaccines.

BACKGROUND OF THE INVENTION

A vaccine is a biological material or preparation that induces animmunologically mediated resistance to certain diseases uponadministration to a subject. Vaccines have been widely used for the past200 years in fighting against infectious diseases and non-infectiousdiseases.

Vaccines comprise an immunogen, which is an antigen that is capable ofinducing humoral and/or cell-mediated immune response of the subject.Antigen presenting cells, including macrophages and other cells of themononuclear phagocyte system actively phagocytose antigen particles andplay a central role in the immune response. Macrophages are cells withinthe tissues that are derived from monocytes. These monocytes/macrophagesphagocytose microbes are then digested to smaller antigenic portions inthe lysosome/phagosome. The resultant antigens are cycled back to thesurface for presentation to the humoral and cellular arms of the immunesystem. Accordingly, monocytes/macrophages are of particular interestbecause they play an important role in both nonspecific and specificdefenses in the host against pathogens.

Dendritic cells are also antigen presenting cells that express MHC classI and class II molecules. In addition to the conventional dendriticcells, dermal dendritic cells are important members of the skin immunesystem. This is because dermal dendritic cells bear high amounts of MHCclass II molecules and therefore can serve as very potent antigenpresenting cells.

An ideal vaccine mimics the rapid uptake and transfer of pathogenicstructures without actually establishing an infection and withoutcausing suppression of the MHC class I pathway.

Recently, many studies have focused on targeted delivery of biologicalmaterials to a cell of monocytic origin to improve therapeutic effectsof the biological materials. It was reported that many vehicles,including microspheres/microparticles, liposomes, nanoparticles,dendrimers, niosomes, and carbon nanotubes could be used for thispurpose. It is desirable to achieve sustained delivery, extendedduration of action, reduced dose and adverse side effects, and improvedpatient compliance with this new delivery approach. Jain et al., ExpertOpin. Drug Deliv.10(3): 353-367 (2013).

Yeast cell wall particles became one of the preferred delivery vehiclesbecause of the hollow, porous microsphere structure formed by the glucanshell derived from a natural source, yeast. Soto et al., Journal of DrugDelivery 2012 (2011). Yeast cell wall particles were used in deliveringvarious substances, such as nucleic acids, proteins, and imagingreporters. See, for example, Bioconjug. Chem. 19(4): 840-848 (2008); andFigueiredo et al., Chemical Communications 47: 10635-10637 (2011).

There remains a need in the art to improve immunization by efficientlydelivering vaccines comprising exogenous proteins, epitopes, antigens,peptides, and/or nucleic acids for MHC presentation with only a very lowamount of exogenous material. In addition, there is a need in the art toprovide yeast cell wall particles for delivering biological materials toimprove delivery efficiency, and to reduce the amount of biologicalmaterials to achieve the same or increased level of efficacy thattargets cells of monocytic origin. The present invention satisfies thisneed.

SUMMARY OF THE INVENTION

In one aspect, the present invention relate to a composition fordelivering a vaccine, comprising (i) a particle and (ii) an exogenousbiological material such as a protein or a fragment thereof, nucleicacid, carbohydrate, tumor lysate, or a combination thereof, loadedwithin the particle. In specific embodiments, the vaccine comprises anantigen that induces an immune response upon administration to asubject. Preferably, the antigen or a fragment thereof is ultimatelypresented on a class I MHC molecule or a class II MHC molecule.

In some embodiments, the protein or fragment thereof, or nucleic acid isselected from the group consisting of a protein, a peptide, an epitope,an antigen, DNA, RNA, cDNA, and an immunogenic fragment or a subunitthereof. In other embodiments, the vaccine is a live vaccine, a killedvaccine, or an attenuated vaccine. In yet other embodiments, the vaccineis a recombinant vaccine.

In some embodiments, the particle is a digestable or biodegradableparticle. Particles suitable for this invention are either synthetic orfrom a natural source, having a hollow inside or a porous structure.Exemplary particles include yeast cell wall particles.

In some embodiments, the loaded particle is incubated with an isolateddendritic cell for about 5 minutes, about 10 minutes, about 15 minutes,about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes,about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes,or about 1 hour, prior to administration. Preferably, the dendritic cellis an immature cell that has been isolated for no more than 8 days. Inother embodiments, the loaded particle is administered without priorincubation with a dendritic cell population.

In some embodiments, the composition of the present invention furthercomprises one or more adjuvants, excipients, and/or preservatives. It iswithin the purview of one of ordinary skill in the art to selectsuitable adjuvants, excipients and/or preservatives for a particularvaccine.

In a preferred embodiment, a small amount of one or more immune responseenhancing adjuvants is added to the composition. The addition of one ormore adjuvants increases the immunogenic effects of the vaccine.Commonly used adjuvants include but are not limited to proteins,peptides, nucleic acids and carbohydrates. Exemplary adjuvants includebut are not limited to monophosphoryl lipid A, CpG ologonucleotides(such as CpG DNA), Poly I:C, Poly ICLC, potent MEW II epitope peptides,beta glucan, and dendritic cell stimulating cytokines such as IL-12 andIFN-γ, as well as DC maturing cytokines such as IL-4 and GM-CSF.Suitable adjuvants are those molecules known to mature DC and interactwith receptors on dendritic cells in order to activate dendritic cellsand further stimulate a more robust generation of T cells, such as CD4+and CD8+ T cells.

In one embodiment, the amount of one or more immune response enhancingadjuvants is at least about 10 ng, at least about 50 ng, at least about100 ng, at least about 200 ng, at least about 300 ng, at least about 400ng, at least about 500 ng, at least about 600 ng, at least about 700 ng,at least about 800 ng, at least about 900 ng, at least about 1 μg, atleast about 5 μg, at least about 10 μg, at least about 15 μg, at leastabout 20 μg, at least about 25 μg, at least about 30 μg, at least about35 μg, at least about 40 μg, at least about 45 μg, at least about 50 μg,at least about 60 μg, at least about 70 μg, at least about 80 μg, atleast about 80 μg, at least about 90 μg, or at least about 100 μg. Inone embodiment, the amount of adjuvant represents between 1-10% of thecomposition. The amount of adjuvant is sufficient to stimulatereceptors, such as the toll-like receptor, on the dendritic cell.

In a related aspect, the present invention relates to a method forefficient delivery of a vaccine to a subject comprising directlyadministering to the dermis of the subject a composition comprising (i)a particle and (ii) an exogenous protein or a fragment thereof, nucleicacid, or a combination thereof, loaded within the particle, as disclosedabove. The dermal dendritic cells phagocytose the loaded particle,thereby triggering the immune response to the vaccine.

In yet another related aspect, the present invention relates to a methodfor producing an incubated dendritic cell containing a biologicalmaterial loaded particle comprising: (i) loading a biological materialinto the particle to produce the loaded particle; (ii) freeze-drying thebiological material loaded particle; and (iii) incubating the biologicalmaterial loaded particle with a dendritic cell, wherein the biologicalmaterial comprises a protein or a fragment thereof, nucleic acid, or acombination thereof, and wherein incubating the loaded particle with thedendritic cell causes the dendritic cell to phagocytose the loadedparticle.

In specific embodiments, the foregoing method further comprises (a)resuspending the biological material loaded particle in solution and (b)freeze-drying the resuspended solution before step (iii). The biologicalmaterial comprises a protein or a fragment thereof, nucleic acid, or acombination thereof.

In specific embodiments, step (iii) comprises: (a) adding a biologicalmaterial into a yeast cell wall particle, (b) incubating the yeast cellwall particle, (c) freeze-drying the yeast cell wall particle and (d)washing the yeast cell wall, wherein the biological material comprises aprotein or a fragment thereof, nucleic acid, or a combination thereof,and wherein steps (b)-(c) are repeated at least once with a step ofadding water into the yeast cell wall particle before step (b) isrepeated.

In specific embodiments, step (iii) comprises: (a) contacting thevaccine loaded particle and the dendritic cell at a ratio from about 1:1to about 100:1, including about 1:1, about 10:1, about 20:1, about 30:1,about 40:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1,and about 100:1; (b) incubating the vaccine loaded particle with thedendritic cell for 1 to 2 hours and (c) collecting the dendritic celland washing the cell.

Furthermore, the present invention relates to a method for preventingand treating infectious diseases and noninfectious diseases, comprisingadministering a composition comprising (i) a particle and (ii) anexogenous biological material loaded within the particle, wherein thebiological material comprises a protein or a fragment thereof, a nucleicacid, or a combination thereof, as disclosed above.

In specific embodiments, the infectious diseases include but are notlimited to virally-mediated, bacterially-mediated, or parasitic diseasescurrently susceptible to vaccine stimulated protective immune responsesor those marginally susceptible with current vaccine technology thatcould be improved with the current invention. In other embodiments, thenon-infectious diseases include but are not limited to cancer bygenerating similar protective immune responses against known and unknownimmunogenic tumor-associated antigens.

In another aspect, the present invention relates to a compositioncomprising a yeast cell wall particle and silicate, wherein the yeastcell wall particle is modified by “capping” with the silicate. In someembodiments, the composition further comprises an exogenous biologicalmaterial loaded within the yeast cell wall particle. In otherembodiments, a composition encompassed by this invention comprises ayeast cell wall particle loaded with a biological material, andoptionally with one or more adjuvants. Preferably, the one or moreadjuvants are loaded within the yeast cell wall particle. Additionally,suitable excipients and/or preservatives can be included in thecompositions of the invention. It is within the purview of one ofordinary skill in the art to select suitable adjuvants, excipientsand/or preservatives. Preferably, the silicate is any organic moietyattached to each of the four oxygen compounds of an orthosilicate, suchas tetraethylorthosilicate (TEOS), tetramethylorthosilicate,tetrapropylorthosilicate, or tetrabutylorthosilicate.

In some embodiments, the biological material includes, but is notlimited to, a specific protein or a fragment thereof, nucleic acid,carbohydrate, tumor lysate, or a combination thereof. The protein or afragment thereof, or nucleic acid is selected from the group consistingof a protein, a peptide, an epitope, an antigen, DNA, RNA, cDNA, and animmunogenic fragment or a subunit thereof.

In some embodiments, a small amount of one or more immune responseenhancing adjuvants is also loaded within the yeast cell wall particleor administered with the loaded YCWP. The addition of one or moreadjuvants to the interior of the YCWP increases the immunogenic effectsof the composition. Commonly used adjuvants include, but are not limitedto, small molecule compounds, proteins, peptides, nucleic acids andcarbohydrates. Suitable adjuvants are those molecules known to maturedendritic cells and interact with receptors on dendritic cells in orderto activate dendritic cells and further stimulate a more robustgeneration of T cells, such as CD4+ and CD8+ T cells. Exemplaryadjuvants include, but are not limited to, monophosphoryl lipid A, CpGoligonucleotides (such as CpG DNA), Poly I:C, Poly ICLC, potent WIC IIepitope peptides, beta glucan, and dendritic cell stimulating cytokinessuch as IL-12, IL-15 and IFN-γ, imiquimod, as well as DC maturingcytokines such as IL-4 and GM-CSF.

In one embodiment, the amount of one or more immune response enhancingadjuvants is at least about 10 ng, at least about 50 ng, at least about100 ng, at least about 200 ng, at least about 300 ng, at least about 400ng, at least about 500 ng, at least about 600 ng, at least about 700 ng,at least about 800 ng, at least about 900 ng, at least about 1 μg, atleast about 5 μg, at least about 10 μg, at least about 15 μg, at leastabout 20 μg, at least about 25 μg, at least about 30 μg, at least about35 μg, at least about 40 μg, at least about 45 μg, at least about 50 μg,at least about 60 μg, at least about 70 μg, at least about 80 μg, atleast about 80 μg, at least about 90 μg, or at least about 100 μg. Inone embodiment, the amount of adjuvant represents between 1-10% (w/w) ofthe composition. The amount of the adjuvant(s) is sufficient tostimulate receptors, such as the toll-like receptor, on the dendriticcell.

In a related aspect, the present invention relates to a method forpreparing a composition comprising a yeast cell wall particle andsilicate. The method comprises contacting a yeast cell wall particlewith a silicate in the presence of ammonia such that the yeast cell wallparticle is “capped” by the silicate. Preferably, the silicate istetraethylorthosilicate (TEOS), tetramethylorthosilicate,tetrapropylorthosilicate, or tetratbutylorthosilicate.

In another related aspect, the present invention relates to a method forefficient delivery of a biological material to a subject comprisingadministering to the subject a composition comprising (i) a yeast cellwall particle capped with silicate; and (ii) a biological materialloaded within the particle. Preferably, the composition is directlyadministered to the dermis of the subject such that the dermal dendriticcells phagocytose the loaded particle, thereby triggering the immuneresponse to the biological material. Cells of monocytic originphagocytose the composition comprising the yeast cell wall particlesloaded with a biological material and capped with silicate, therebypromoting differentiation into mature dendritic cells for proper antigenpresentation.

In yet another related aspect, the present invention relates to a methodfor producing a cell mixture containing a yeast cell wall particleloaded with a biological material and capped with a silicate comprising:(i) loading a biological material into a yeast cell wall particle toproduce a loaded particle; (ii) capping the loaded particle with asilicate, (iii) freeze-drying the capped, loaded particle; and (iv)incubating the capped, loaded particle with a cell of monocytic origin,such as a pre-dendritic cell, a dendritic cell or a partiallydifferentiated dendritic cell, wherein the incubation causes the cell ofmonocytic origin to phagocytose the capped, loaded particle. In someembodiments, the cell of monocytic origin is a dendritic cell, and thebiological material includes, but is not limited to, a specific proteinor a fragment thereof, nucleic acid, carbohydrate, tumor lysate, or acombination thereof.

In specific embodiments, the loading step of the foregoing method forproducing an dendritic cell containing a capped, loaded yeast cell wallparticle comprises: (a) suspending a yeast cell wall particle and abiological material in a diluent and incubating for a period of time,such as about two hours, to allow the biological material to be absorbedby the yeast cell wall particle and (b) freeze-drying the suspension toload the biological material within the yeast cell wall particle. Ifnecessary, steps (a) and (b) are repeated at least once to increase theloading efficiency.

In specific embodiments, the incubating step of the foregoing method forproducing an isolated dendritic cell containing a capped, loaded yeastcell wall particle further comprises: (a) contacting the capped, loadedparticle with a dendritic cell at a ratio from about 1:1 to about 100:1,including about 1:1, about 10:1, about 20:1, about 30:1, about 40:1,about 50:1, about 60:1, about 70:1, about 80:1, about 90:1, and about100:1; (b) incubating the capped, loaded particle with the dendriticcell for 1 to 2 hours and (c) collecting the dendritic cell and washingthe cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a process for producing dendritic cells.

FIG. 2 depicts a process for producing tumor lysate.

FIG. 3 depicts a process for producing yeast cell wall particles.

FIG. 4 depicts a process for loading biological material into yeast cellwall particles.

FIG. 5 depicts a process for producing vaccine particle loaded dendriticcells.

FIG. 6A depicts the lungs of unvaccinated mice 21 days followinginoculation with one million B16 tumor cells IV (dark spots are melanomametastases). FIG. 6B depicts the lungs of vaccinated mice 21 daysfollowing inoculation with one million B16 tumor cells IV, which micewere vaccinated with tumor lysate and yeast cell wall particles simplymixed 3 days prior to tumor challenge. FIG. 6C depicts the lungs ofvaccinated mice 21 days following inoculation with one million B16 tumorcells IV, which mice were vaccinated with yeast cell wall particlesloaded with tumor lysate 3 days prior to tumor challenge.

FIGS. 7A-7E depict the structure of the silicate capped yeast cell wallparticles. Silicon is depicted in the darker grey color while thelighter grey color represents carbon. FIG. 7A shows the structure oftetraethylorthosilicate (TEOS). FIG. 7B shows the partial hydrolysisproducts of TEOS initiated by the ammonia in the reaction mixture(ethylorthosilicate) forming H-bonds between their OH groups. FIG. 7Cshows the silanol condensation product resulting from the loss of waterfrom the H-bonded ethylorthosilicate molecules. This reaction continuesin developing polymeric silicates. FIG. 7D structural group shows thesimilar H-bonding between the polymeric silicate structures and theprimary hydroxyl group of the β-glucan bonding between the polymericsilicate structures and the primary hydroxyl group of the β-glucanstructure of the yeast cell wall particles. FIG. 7E shows the resultingcovalent bond formation between the polymeric silicates and the yeastcell wall particles resulting from this silanol condensation and causingthe capping of the loaded yeast cell wall particles.

FIG. 8 depicts the percentage of survival of each group of the mice:“control” group that received IV injection of B16 melanoma tumor cellsonly; “regular YCWP” group that received IV injection of B16 melanomatumor cells and interdermal injection of uncapped YCWPs loaded with B16tumor lysate; “Si capped YCWP” group that received IV injection of B16melanoma tumor cells and interdermal injection of silicate capped YCWPsloaded with B16 tumor lysate; “regular YCWPs+AD” group that received IVinjection of B16 melanoma tumor cells and interdermal injection ofuncapped YCWPs loaded with B16 tumor lysate and adjuvants including CpGoligonucleotide and monophosphoryl lipid A, and “Si capped YCWPs+AD”group that received IV injection of B16 melanoma tumor cells andinterdermal injection of silicate capped YCWPs loaded with B16 tumorlysate and adjuvants including CpG oligonucleotide and monophosphoryllipid A.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Reference is made herein to various methodologies known to those ofordinary skill in the art. Publications and other materials settingforth such known methodologies to which reference is made areincorporated herein by reference in their entireties as though set forthin full.

Definitions

The term “about” in connection with numerical values and ranges meansthat the number comprehended is not limited to the exact number setforth herein, and is intended to refer to ranges substantially withinthe quoted range while not departing from the scope of the invention. Asused herein, “about” will be understood by persons of ordinary skill inthe art and will vary to some extent on the context in which it is used.For example, “about” means that +/−10% of a particular numerical valuefollowing the term.

As used herein “subject” or “patient” denotes any animal in need oftreatment with a vaccine. For example, a subject may be suffering fromor at risk of developing a condition that can be treated or preventedwith a vaccine. As used herein “subject” or “patient” includes humans.

As used herein, the phrases “therapeutically effective amount” and“therapeutic level” mean that the vaccine dosage or plasma concentrationof the compositions described herein in a subject, respectively, thatprovides the specific response for which the biological material orvaccine is administered in a subject in need of such treatment. Forconvenience only, exemplary dosages, delivery amounts, therapeuticallyeffective amounts and therapeutic levels are provided below withreference to adult human subject. Those skilled in the art can adjustsuch amounts in accordance with standard practices as needed to treat aspecific subject and/or condition/disease.

As used herein, the term “capping” or “capped” means that a polymericstructure, like a “mesh net,” covers or coats the yeast cell wallparticle such that the biological material loaded within the yeast cellwall particle is retained or entrapped therein. The polymeric structurecan be formed by a silicate, such as a tetraalkylorthosilicate.

Vaccine

The term “immunization” means a process by which a subject becomesprotected against a particular condition, disease or diseases, usuallyby receiving a vaccine.

The term “vaccine” is a biological material or product that inducesimmune response in the body of a subject upon administration, e.g., byinjection, by oral administration, or by aerosol administration. Vaccinecomprises at least one active component, such as an antigen that inducesimmune response, and additional components such as adjuvants,conjugates, preservatives, and other excipients including diluents,stabilizers, etc.

Exemplary antigens useful in vaccine applications include allergens,viral antigens, bacterial antigens and antigens derived from parasites.For preventing and treating infectious diseases, bacterial antigens andviral antigens are preferred. Suitable viral antigens include HIV, EBV,HBV, HCV, CMV, and Herpes virus. Additionally, toxins (usually producedby bacteria) can be used as antigens. For non-infectious diseases suchas cancer, preferred antigens include tumor associated antigens, withwhich the artisan will be familiar (e.g., carcinoembryonic antigen,prostate-specific membrane antigen, melanoma antigen, adenocarcinomaantigen, leukemia antigen, lymphoma antigen, sarcoma antigen, MAGE-1,MAGE-2, MART-1, Melan-A, p53, gp 100, antigen associated with coloniccarcinoma, antigen associated with breast carcinoma, like HER2 andmammoglobin A, Mucl, Trp-2, telomerase, PSA and antigen associated withrenal carcinoma), and can include a combination of antigens or antigenicfragments. In one embodiment, the particle is loaded with tumor celllysate.

Biological Material

The biological material encompassed by this invention includes, but isnot limited to, a specific protein or a fragment thereof, nucleic acid,carbohydrate, tumor lysate, or a combination thereof. One of ordinaryskill in the art would understand that fragments of a protein, e.g. apeptide of any length, an epitope, or a subunit of a protein, whichproduce immunogenic response of a subject upon administration can beused.

In recent years, nucleic acids such as DNA, RNA, cDNA or fragmentsthereof, are also used as vaccines. In general, the DNA is extractedfrom an infectious agent's DNA, and then modified/enhanced by geneticengineering before delivering to a subject by electroporation, gene gun,etc.

The biological material of the present invention may be live, wild-typepathogens. Preferably, and antigens are in inactivated or attenuatedforms, such as killed viruses, pieces of bacteria, and subunits orimmunogenic functional fragments of proteins, polypeptides or nucleicacids. More preferably, the biological material does not cause illnessbut can effectively provoke an immune response of the subject andprotects the subject against future infection of a particular disease.

It is to be understood that yeast cell wall particles have a pore sizeof at least about 30 nm, and therefore, any molecule/object with aradius of rotation of 30 nm or less can be loaded within the yeast cellwall particles. For example, some viruses or viral particles having asize less than 30 nm (e.g., tobacco mosaic virus) can be loaded withinyeast cell wall particles, as well as other antigens, including tumorlysate.

Adjuvants

A number of immune response enhancing agents can be added to thecomposition as adjuvants to boost immune response such that when thecomposition is administered to a subject, for example, directly to thedermis of the subject, the immune response is boosted by the adjuvantscomparing to administering a composition without any adjuvant.Alternatively, when the composition comprising a biological materialloaded particle is incubated with a dendritic cell, the adjuvantsexhibit an increased effect on the dendritic cell while dramaticallydecreasing any systemic effects from such adjuvants. The biologicalmaterial comprises a protein or a fragment thereof, nucleic acid,carbohydrate, tumor lysate, or a combination thereof.

A number of immune response enhancing agents can be added to thecomposition for loading within the yeast cell wall particle, asadjuvants to boost immune response such that when the composition isadministered to a subject, for example, directly to the dermis of thesubject, the immune response is boosted by the adjuvants compared toadministering the composition without any additional adjuvants. Forexample, when the yeast cell wall particles are loaded with a biologicalmaterial, and the loaded yeast cell wall particle are also incubatedwith a dendritic cell, and the adjuvants exhibit an increased effect onthe dendritic cell while dramatically decreasing any localized orsystemic effects from such adjuvants.

It is within the purview of one of ordinary skill in the art to selectone or more suitable adjuvants for this invention. For instance,monophosphoryl lipid A, CpG oligonucleotides, Poly I:C, Poly ICLC,potent MHC II epitope peptides, and dendritic cell stimulating cytokinessuch as IL-12, IL-2, and GM-CSF are good adjuvant candidates of thisinvention.

Suitable adjuvants are those molecules known to mature dendritic cellsand interact with receptors on dendritic cells in order to activatedendritic cells and further stimulate a more robust generation of Tcells, such as CD4+ and CD8+ T cells. For instance, monophosphoryl lipidA, CpG oligonucleotides, Poly I:C, Poly ICLC, potent MHC II epitopepeptides, and dendritic cell stimulating cytokines such as IL-12, IL-2,and GM-CSF, small molecules such as imiquimod are good adjuvantcandidates of this invention.

Particle

As described herein, “particle” refers to any hollow and porousstructure that can contain vaccine therein and also allow the vaccine toexit the structure. In some embodiments, the size of the particle isabout 0.5 to about 5 μm, which approximates the size of bacterium toallow the particle to be ingested by monocytes, such as dendritic cells.In specific embodiments, the size of the particle is about 0.5 to about1 μm. In specific embodiments, the size of the particle is about 0.5 toabout 2.5 μm. In some embodiments, the particle can be any particle witha glycan network, so long as the particle is about 0.5 to about 5 μm insize.

Preferably, the particle is a digestable or biodegradable particle. Insome embodiments, the particle is not limited by a particular shape ormaterial, but can be any shape, size, or material having a hollow orporous structure that allows the particle to be phagocytosed bymonocytes, including dendritic cells.

Yeast Cell Wall Particles

In another embodiment, the particle is a yeast cell wall particle YCWP,which is prepared from yeast cell wall such that the particle has ahollow or porous structure to encapsulate a biological material therein.The biological material comprises a protein or a fragment thereof,nucleic acid, or a combination thereof. In one embodiment, the YCWP isprepared from Saccharomyces cerevisiae. In another embodiment, the YCWPapproximates the size of microbial structures that cells of themononuclear phagocyte system and other phagocytic cells typicallyingest. In specific embodiments, the YCWP is about 1-25 μm, preferably1-5 μm, 5-10 μm, 10-15 μm, 15-20 μm, 15-25 μm, or 20-25 μm. For example,the YCWP is about 20 μm.

In one embodiment, the YCWP is prepared by (a) suspending yeast toproduce a suspension, (b) incubating the suspension, (c) centrifugingthe suspension and removing the supernatant and (d) recovering theresulting YCWP. In another embodiment, steps (a)-(d) are repeated atleast 1, 2, 3 or 4 times.

In another embodiment, the YCWP is prepared by (a) suspending yeast in asolution to produce a first suspension, (b) incubating the firstsuspension, (c) centrifuging the first suspension and removing thesupernatant, (d) suspending the resulting pellet to produce a secondsuspension, (e) incubating the second suspension, (f) centrifuging thesecond suspension and removing the supernatant and (g) washing theresulting pellet to recover the YCWP. In another embodiment, the YCWP issterilized.

In specific embodiments, the yeast is suspended in NaOH, including 1MNaOH. In specific embodiments, the first suspension is incubated atabout 80° C. for about 1 hour or for 1 hour. In specific embodiments,the centrifuging is performed at about 2000 times gravity for about 10minutes, or at 2000 times gravity for 10 minutes. In specificembodiments, the pellet is suspended in water, including water at aboutpH 4.5 or at pH 4.5. In specific embodiments, the second suspension isincubated at about 55° C. for about 1 hour or at 55° C. for 1 hour. Inspecific embodiments, the pellet is washed in water at least 1, 2, 3 or4 times. In specific embodiments, the pellet is washed once.

In another embodiment, the YCWP is sterilized using isopropanol and/oracetone following washing of the pellet. In specific embodiments, otherknown alcohols are appropriate. In specific embodiments, the YCWP isallowed to fully dry after sterilization. In another embodiment, theYCWP is resuspended after being allowed to dry. In specific embodiments,the YCWP is resuspended in PBS, such as 1X PBS.

In another embodiment, the YCWP is allowed to dry and then to be frozenbefore the biological material is loaded into the YCWP and/or beforecapped with silicate, in order to place the YCWP in storage before use.In specific embodiments, the YCWP is freeze dried and stored at about 4°C. or lower. In specific embodiments, the YCWP is freeze dried andstored at 4° C. The biological material comprises a specific protein ora fragment thereof, nucleic acid, carbohydrate, tumor lysate, or acombination thereof.

Biological Material Loaded Particle

The particle, for example, the yeast cell wall particle, is loaded witha biological material, such as a specific protein or a fragment thereof,nucleic acid, carbohydrate, tumor lysate, or a combination thereof. Inone embodiment, the biological material is loaded into the particle byincubating the biological material and a suspension of particle, forexample, the yeast cell wall particles together and allowing thebiological material to penetrate into the hollow insides of theparticles.

In another embodiment, after the particle or the yeast cell wallparticle is incubated or loaded with the biological material, thecombination is freeze-dried to create an anhydrous vaccine within theparticle. By freeze-drying, the biological material is trapped withinthe particle and ready to be phagocytosed by a monocyte, such as adendritic cell. In specific embodiments, the freeze-drying is the onlymechanism used to trap the biological material within the particle. Inspecific embodiments, the entrapment is not caused by a separatecomponent blocking the biological material from exiting the particle,for example, by physical entrapment, hydrophobic binding, any otherbinding. In specific embodiments, the entrapment is not caused bycrosslinking or otherwise attaching the biological material to theparticle outside of any attachment that may occur upon freeze-drying. Inspecific embodiments, the compositions of the present invention do notinclude any additional component that specifically assists in evadingthe lysosome. The biological material includes, for example, a specificprotein or a fragment thereof, nucleic acid, carbohydrate, tumor lysate,or a combination thereof.

In another embodiment, the biological material is incorporated into theyeast cell wall particle. In specific embodiments, the number of YCWPsis about 1×10⁹ and the volume of biological material is about 50 μL. Inspecific embodiments, the incubation is for about one hour or less thanone hour at about 4° C. In some embodiments, the combination of YCWPsand biological material is freeze dried over a period of less than orabout 2 hours.

In another embodiment, the loaded yeast cell wall particle is cappedwith a silicate. Specifically, in some embodiments the loaded YCWPs arecapped by contacting the YCWPs with a silicate, such astetraalkylorthosilicate, in the presence of ammonia, such that theloaded YCWPs are capped with the silicate. In preferred embodiments, theloaded YCWPs are capped with the silicate within about 60 minutes, about45 minutes, about 30 minutes, about 15 minutes, about 10 minutes, about5 minutes or about 2 minutes. The reactivity of thetetraalkylorthosilicates is such that under hydrolysis mediated by theammonia, the tetraalkylorthosilicates react with the primary hydroxylsof the β-glucan structure of the YCWPs. The tetraalkylorthosilicatesalso self-react with the ends of these cell wall silicates to form“bridges” such as —O—Si(OH)₂—O— or in three dimensions such as—O—Si(—O—Si—O—) (OH)—O— or —Si(—O—Si—O—)₂—O—. These bridges may occuracross the pores in the YCWPs such that the retention of the loaded drugor biological material therein is increased. The structure of the cappedYCWPs is depicted in FIG. 7E. Such a capped, loaded YCWP can be freezedried.

The inventor of the present application unexpectedly discovered thatloaded YCWPs capped with silicate are an effective vaccine deliverysystem. More specifically, the capped YCWPs retain more loaded materialthan the uncapped YCWPs. Even more surprisingly, the capped YCWPs notonly deliver significantly more released biological material into thecytoplasm of the phagocytic cells but also deliver significantly moreloaded particles into the phagocytic cells in comparison to the uncappedYCWPs, as detailed in the working examples.

In another embodiment, the loaded particle is resuspended in a diluentor solution after the freeze-drying. In specific embodiments, thediluent or solution is water. In specific embodiments, the loadedparticle is resuspended and/or incubated with additional biologicalmaterial, for example, vaccine, to penetrate the particle and thecombination is then freeze-dried again. In other embodiments, thecombination is subjected to multiple freeze-drying and resuspensions. Inother embodiments, the biological material loaded particle is sterilizedin ethanol after the freeze-drying and before use. The biologicalmaterial includes, for example, a protein or a fragment thereof, nucleicacid, carbohydrate, tumor lysate, or a combination thereof.

In specific embodiments, the biological material is loaded into theparticle by (a) incubating the biological material and a suspension ofthe particles, allowing the biological particle to penetrate into thehollow insides of the particles and freeze-drying the suspension ofloaded particle and (b) optionally resuspending the particles,incubating the resuspended particles and freeze drying the resuspendedparticles and any vaccine not already in the particle.

In specific embodiments using YCWPs, the number of YCWPs is about 1×10⁹and the volume of the biological material is about 50 μL. In specificembodiments, the number of YCWPs is 1×10⁹ and the volume of thebiological material is 50 μL. In specific embodiments, the incubation instep (a) is for less than one hour at about 4° C. In specificembodiments, the incubation in step (a) is for about one hour at 4° C.In some embodiments, the foregoing suspension is freeze dried in step(a) over a period of less than 2 hours or over a period of about 2hours. In some embodiments, the YCWPs in step (b) are resuspended inwater, including about 50 μL of water or 50 μL of water. In someembodiments, the resuspended YCWPs are incubated in step (b) for lessthan or about one hour at about 4° C. or for less than or about 2 hoursat 4° C. The biological material includes a specific protein or afragment thereof, nucleic acid, carbohydrate, tumor lysate, or acombination thereof.

Prior to administration, the capped, loaded yeast cell wall particle isresuspended in a pharmaceutically acceptable excipient, such as PBS or asaline solution.

Dendritic Cell

As described herein, “dendritic cell” refers to a cell generated from aperipheral blood mononuclear cell (“PBMC”). In one embodiment, adendritic cell is prepared by (a) collecting blood, (b) diluting theblood, (c) performing a density gradient separation of PBMCs, (d) lysingred blood cells and washing the PBMCs, (e) incubating the PBMCs, (f)removing nonadherent cells and (g) culturing adherent cells in media.

In some embodiments, the dendritic cell is an immature dendritic cellthat has been cultured for no more than 5 days. In other embodiments,the dendritic cell has been cultured for 6-8 days.

In specific embodiments, the blood is heparinized. In specificembodiments, the density gradient separation at step (c) comprisesplacing the blood in a Lymphocyte Separation Medium and thencentrifuging the blood. In specific embodiments, the centrifuging isperformed at about 1000 times gravity for about 20 minutes or at 1000times gravity for 20 minutes. In specific embodiments, a secondcentrifuging is performed before step (d) and is performed at about 500g for about 5 minutes or is performed at 500 g for 5 minutes. Inspecific embodiments, a third centrifuging is performed before step (d)and is performed at about 500 g for about 10 minutes or is performed at500 g for 10 minutes. In specific embodiments, the centrifuging isperformed at about 1200 times gravity for about 10 minutes or at 1200times gravity for about 15 minutes. In specific embodiments, a secondcentrifuging is performed before step (d) and is performed at about 500g for about 5 minutes or is performed at 500 g for 5 minutes. Inspecific embodiments, the lysing is performed using an ACK lysingsolution, followed by incubation, preferably at room temperature forabout 5 minutes, and followed by a subsequent centrifugation. Inspecific embodiments, the PBMCs are washed in RPMI medium. In specificembodiments, the PBMCs are incubated at step (e) in flasks at about 37°C. for about 1-2 hours or at 37° C. for 1-2 hours. In specificembodiments, serum-free DC media is added to the flask.

In some embodiments, one or more cytokines is present in the culturemedia, including, but not limited to, granulocyte macrophage colonystimulating factor (e.g., 800 units/ml) and IL-4 (e.g., 500 units/ml).

Vaccine Compositions

In some embodiments, the biological material loaded particles aredirectly injected into the dermis of a subject such that the loadedparticles are phagocytosed by dermal dendritic cells. In someembodiments, optionally, the biological material loaded particle is invitro phagocytosed within a monocyte, preferably a dendritic cell. Insome embodiments, the yeast cell wall particles loaded with a biologicalmaterial and capped with silicate are directly injected into the dermisof a subject such that the particles are phagocytosed by dermaldendritic cells. In one embodiment, the biological material loadedparticle is incubated with a dendritic cell such that the cellphagocytoses the biological material loaded particle. The biologicalmaterial includes, for example, a specific protein or a fragmentthereof, nucleic acid, carbohydrate, tumor lysate, or a combinationthereof. In other embodiments, the capped, loaded particles arephagocytosed by a monocyte in vitro, wherein the monocyte is preferablya dendritic cell.

In specific embodiments, the particle is incubated with the dendriticcell at a ratio of from about 1:1 to about 100:1, optionally prior tohuman administration. The incubation can be performed for about 1 hour,1 hour or preferably less than 1 hour.

In specific embodiments, the dendritic cell containing the capped,loaded particle is collected and washed, for example, at least 1, 2, 3or 4 times. In other embodiments, the dendritic cells are incubatedafter washing, resuspended in freezing medium, and frozen for storagebefore use. In specific embodiments, the resuspension produces aconcentration of about 10×10⁶ cells per ml or 10×10⁶ cells per ml. Inspecific embodiments, the resuspension is frozen for storage before use.

Formulation

The compositions of the present invention may be formulated for mucosaladministration (e.g., intranasal and inhalational administration) or forpercutaneous administration. The composition of the invention can alsobe formulated for parenteral administration (e.g., intramuscular,intravenous, or subcutaneous injection), and injected directly into thepatient and target cells of monocytic origin, like macrophages anddendritic cells. In specific embodiments, the capped, biologicalmaterial loaded particles without prior incubation with dendritic cellsare directly injected into the dermis of a subject. Thus, thecompositions of the present invention may be administered just like aconventional vaccine. This also substantially reduces cost because ofthe lower level of skill required In other embodiments, the capped,loaded particle is first incubated with cells of monocytic origin, suchas dendritic cells, prior to administration to a subject.

Formulations for injection may be presented in unit dosage form, e.g.,in ampules or in multi-dose containers, optionally with an addedpreservative. The compositions may take such forms as suspensions,solutions or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilizing and/or dispersingagents. The composition of the present invention may also be formulatedusing a pharmaceutically acceptable excipient. Such excipients are wellknown in the art, but typically will be a physiologically tolerableaqueous solution. Physiologically tolerable solutions are those whichare essentially non-toxic. Preferred excipients will either be inert orenhancing, but a suppressive compound may also be used to achieve atolerogenic response. Alternatively, the composition is not administeredwith any other immunosuppressive treatment, such as steroids orchemotherapy.

Therapeutic Methods

The compositions of the present invention attract phagocytic cells, suchas cells of the mononuclear phagocyte system, including monocytes,macrophages, dendritic cells or immature dendritic cells and thereforecan be used as a vaccine. In the field of vaccination, cells of themononuclear phagocyte system are considered “professional” antigenpresenting cells and thus, are the ideal target for vaccine delivery. Itis well known that presentation of an antigen within an APC is vastlymore effective in generating a strong cellular immune response thanexpression of this same antigen within any other cell type. Therefore,the ability of the compositions of the present invention to present anantigen on an antigen presenting cell via class I MHC and class II MHCmolecules dramatically enhances the efficacy of such a vaccine.

The present invention contemplates both prophylactic and therapeuticuses of the compositions disclosed herein for infectious diseases suchas virally-mediated, bacterially-mediated, and parasitic diseasescurrently targeted with vaccine strategies or those marginallysusceptible due to limitations of current vaccine technology, andnoninfectious diseases, including cancer. The disease to be treated isnot particularly limiting, but depends on the biological material loadedinto the particle. Such exemplary biological material includes a tumorlysate, protein or a fragment thereof, nucleic acid, carbohydrate, or acombination thereof.

The compositions of the present invention come into contact withphagocytic cells either in vivo or in vitro. Hence, both in vivo and invitro methods are contemplated. As for in vivo methods, the compositionsof the present invention are generally administered parenterally,usually intravenously, intramuscularly, subcutaneously, interdermally orintradermally. They may be administered, e.g., by bolus injection orcontinuous infusion. In in vitro methods, monocytic cells are contactedoutside the body and the contacted cells are then parenterallyadministered to the patient.

Dosage

In some embodiments, about 200 μL of a 10×10⁶ concentration of dendriticcells containing biological material locaded particles, or capped,biological material loaded yeast cell wall particles forms one dose ofthe treatment. In another embodiment, the dose is administered bydiluting the 200 μL aliquot to a final volume of 1 ml beforeadministering the dose to a subject. In specific embodiments, thealiquot is diluted with sterile saline containing 5% human serumalbumin. In specific embodiments, the 200 μL aliquot will need to bethawed before dilution. In such a scenario, the length of time betweenthawing and administration of the dose to a subject will be no longerthan 2 hours. In some embodiments, the diluted aliquot is administeredin a 3 cc syringe. In some embodiments, a syringe needle no smaller than23 gauge is used.

In another embodiment, a subject is administered at least 1, 2, 3 or 4doses of the compositions of the present invention. In specificembodiments, a subject is re-vaccinated once every 4 weeks. In someembodiments, the composition comprising biological material loadedparticle is administered to a subject without first fusing to dendriticcells. In specific embodiments, a subject is re-administered with thecomposition once every 4 weeks. In specific embodiments, about 1-2million dendritic cells containing the biological material loadedparticles or the capped, loaded particles is administered optionally byinjection at each vaccination. In specific embodiments, the biologicalmaterial loaded particles or capped, loaded particles are injected in asubject at or near (1) a site of infection or disease, or (2) a lymphnode. The biological material includes, for example, a protein or afragment thereof, nucleic acid, or a combination thereof.

The vaccine composition can also contain biological adjuvants, includingbut not limited to nucleic acids such as CpG oligonucleotides, proteinsor peptide epitopes such as the tetanus toxoid MHC class II-binding p30peptide.

The present invention is further illustrated by the following workingexamples, which are for illustration purpose only and by no meanslimiting the scope of the present invention.

EXAMPLES Example 1: Preparing Dendritic Cells

Dendritic cells were generated from a patient's PBMCs. PBMCs werecollected from the patient by a blood draw of 200 ml following standardoperating procedures. The blood was then transferred to 250 mlcentrifuge tubes and diluted 1:1 with 1×PBS. Then, 35 ml of the dilutedblood was layered over 15 ml of room temperature Lymphocyte SeparationMedium (LSM; Mediatech) in 50 ml tubes and centrifuged at 1000 g for 20minutes at room temperature. The PBMC layers were removed by pipettingfrom the LSM gradients and placed into clean 50 ml centrifuge tubes.Four volumes of 1×PBS were added and the tubes were inverted to mix thecontents. The PBMCs were then centrifuged at 500 g at room temperaturefor 5 minutes. Ten ml of 1×PBS were added into each tube and the cellswere resuspended and pooled into 1 tube. The PBMCs were againcentrifuged at 500 g at room temperature for 10 minutes, resuspended in20 to 40 ml of ACK lysing solution (Cambrex) and incubated at roomtemperature for 5 minutes. The cells were then centrifuged again at 1500rpm for 5 minutes. The PBMCs were resuspended in 30 ml RPMI-1640 medium(Mediatech). The cells were then transferred into 2-4 T75 flasks. Theflasks were incubated at 37° C. for 1 to 2 hours. The non-adherent cellswere then removed by rinsing. Afterwards, 10 ml of 1×PBS were added intoeach flask, the flask swirled, and the PBS removed. Afterwards, 10 ml ofcomplete DC media (serum-free DC Medium+800 U/ml GM-CSF+1000 U/ml IL-4)was added to each flask. The flasks were then incubated at 37° C., 5%CO2 for 2 days. On Day 3, 10 ml of complete DC medium was added intoeach flask. The cells were then incubated for another 2 days. On Day 6or 7, the resulting immature dendritic cells were ready for use.

FIG. 1 provides an overview of the generation of dendritic cells.

Example 2: Preparing the Antigen

Synthetic antigens such as peptides can be easily produced commerciallyand provided in lyophilized state. These peptides can be re-constitutedand co-incubated with the prepared YCWP for loading. Similarly,recombinant proteins and/or isolated proteins can be suspended insolution and co-incubated with the YCWP for loading as discussed below.

Example 3: Preparing Tumor Lysate

A tumor sample was obtained from a patient. After separating fat andnecrotic tissue away from the tumor tissue, the tissue was weighed and1X PBS added (50 μL of PBS per 200 μg of tissue) and the tumor wasminced thoroughly with scalpels in 1×PBS. The tumor cells were thensubjected to 4 cycles of freeze and thaw. The freezing was performed inliquid nitrogen for 20 minutes and the thawing was performed at roomtemperature. Prepared tumor lysate was quantified by aspectrophotometer. An aliquot was taken for quality control testing. Theremainder was stored at <−135° C. in preparation for vaccinepreparation. Small amounts of adjuvant can optionally be added after thefreeze thaw cycles.

FIG. 2 provides an overview of the tumor cell lysate processing.

Example 4: Preparing Yeast Cell Wall Particles

YCWPs were prepared from Fleishmans Baker's Yeast or equivalent.Briefly, 10 g of Fleishmans Baker's yeast was suspended in 100 ml of 1 MNaOH and heated to 80° C. for one hour. The undissolved yeast cell wallswere recovered by centrifugation at 2000×g for 10 minutes. The recoveredyeast cell walls were then resuspended in 100 ml of water with the pHadjusted to 4.5 with HCl and incubated at 55° C. for an additional hour,and subsequently recovered by centrifugation. The recovered YCWPs werethen washed with water once, isopropanol 4 times and finally acetone 2times. Once the YCWPs were fully dried they were resuspended in PBS,counted, aliquoted into groups of 1×10⁹ particles and freeze dried foruse in manufacturing the vaccine.

FIG. 3 provides an overview of the yeast cell wall particles processing.

Example 5: Preparing Yeast Cell Wall Particles

Three grams of active dry yeast (Fleischmann's or equivalent) werewashed three times in water by suspending the yeast in 30 mL of sterilewater, vortexing, and centrifuging at 800-1000 x g for 5 minutes at roomtemperature. After decanting the supernatant, the yeast pellet wasresuspended in 50 mL of 1 M NaOH and heated in a 90° C. water bath for 1hour.

The yeast suspension was subsequently centrifuged at 800-1000 x g for 5minutes, and the pellet was resuspended in 25-30 mL of acid water (pHadjusted to 4.5 with HCl). The acid water wash step was repeated untilthe pH of the suspension is <7.0. Then the pellet was resuspended in 30mL acid water and incubated in a 75° C. water bath for 1 hour. The yeastpellet was recovered by centrifugation at 1000×g for 5 minutes, and thenwashed with 10 mL of sterile water 3 times, 10 mL of isopropanol 4 timesand finally 10 mL of acetone 2 times. The acetone was carefully removed,and the pellet was spread evenly on the glass surface of a beaker,allowed to air dry overnight.

The dried YWCPs were collected and stored in a vacuum jar at 4° C. andthen washed in 10-15 mL of filtered 70% ethanol 3 times. The YWCPs werebriefly sonicated on the final wash, and the sonication was repeated ifnecessary to disperse clumps. Once the ethanol was removed, the YWCPswere washed in sterile water. Aliquots of 100 μl of YWCPs were dispensedinto 2.0 mL rounded bottom snap top centrifuge tubes, placed in freezerfor 1 hour, freeze dried, and stored in a vacuum jar at 4° C. for futureuse.

Example 6: Preparing Yeast Cell Wall Particles

Yeast cell wall particles (YCWPs) were prepared by suspendingSacharomyces cerevisiae (100 g of Fleishmans Bakers yeast, AB Mauri FoodInc., Chesterfield, Mo.) in 1 L of 1 M NaOH and heating to 80° C. for 1h. The insoluble material containing the yeast cell walls was collectedby centrifugation at 2000×g for 10 min. This insoluble material was thensuspended in 1 L of water, brought to pH 4-5 with HCl, then incubated at55° C. for 1 h. The insoluble residue was again collected bycentrifugation and washed once with 1 L of water, four times with 200 mLof isopropanol, and twice with 200 mL of acetone. The resulting slurrywas dried at room temperature in a sterile hood to produce 12.4 g of afine, slightly off-white powder. The powder composed of dry YCWPs wascarefully weighed and suspended in sterile distilled water at aconcentration of 10 mgs/ml, 1 ml aliquots were placed in sterileEppendorf tubes, frozen at −60° C. and freeze dried at 0.012 mBar.Because the boiling point of isopropanol and acetone is considerablybelow that of water, any possible contamination by these solvents wouldbe removed under these high vacuum conditions.

Example 7: Preparing Tumor Lysate and Loading YCWPs

Tumor protein antigens are released from tumor tissue by three freeze(−60° C.)/thaw cycles followed by centrifugation at 21,000 g to removeall non-soluble material. The soluble tumor antigenic material andoptionally included adjuvant material is loaded into the inside of thehollow YCWPs by two hours of incubation at 4° C. to allow the smallvolume of soluble tumor lysate to fully penetrate the hollow insides ofthe YCWPs. The volume of soluble tumor lysate used is carefullycalculated to closely approximate the volume of the insides of the YCWPssuch that the vast majority of the soluble tumor lysate, afterincubation, resides within the hollow insides of the YCWPs. Followingincubation the fully solvated YCWPs are frozen at −60° C. and all waterremoved by freeze drying at 0.012 mBar vacuum for 8 hours leaving theanhydrous tumor lysate antigenic material mostly inside the hollowYCWPs. In order to drive any residual tumor lysate material into theinsides of the YCWPs the same tiny calculated volume of the insides ofthe YCWPs of sterile water is added to the dried partially loaded YCWPsand again incubated for two hours at 4° C., followed again by freezedrying at 0.012 mBar vacuum for 8 hours.

Example 8: Loading Biological Material into YCWPs

A suspension of fully anhydrous YCWPs (1×10⁹) is placed in contact with50 μL of a peptide in PBS over a period of 2 hours at 4° C., allowingthe peptide to penetrate into the hollow insides of the YCWPs to produceloaded YCWPs. The suspension is then freeze dried for 2 hours. Afterfreeze drying, 50 μL of water is added to the loaded YCWPs, incubatedfor another 2 hours at 4° C. and again freeze dried to yield YCWPs withdry biological material within their hollow insides. The loaded YCWPsare then sterilized by washing in ethanol and maintained in ethanol.

FIG. 4 provides an overview of the YCWPs loading procedure.

Example 9: Loading YCWPs with Tumor Lysate

A patient tumor biopsy sample was mixed carefully with 50-100 μl oflysis buffer (PBS) (depending on the amount of the tumor sample),avoiding bubbles during mixing, and was then incubated at 4° C. for 30minutes. The mixture was subjected to freeze-thaw 3 times in acetone-dryice bath and 37° C. water bath, and centrifuged at 4° C. for 10 minutesat maximum speed. 50 μl of the prepared tumor lysate was added in asterile 2 mL centrifuge tube containing 10 mg of dried YCWPs such thatthe liquid tumor lysate covered the YCWPs. The mixture was incubated at4° C. for 2 hours until the liquid tumor lysate soaked into the YCWPs.

The tube was then placed into a −85° C. freezer for 30 minutes for aquick freeze of the pellet. The tube was placed on freeze drierovernight. 50 μl of sterile water was added onto the dried yeast pelletand incubated at 4° C. for 2 hours to allow the liquid to soak into thepellets.

The tube was placed into a −85° C. freezer for 30 minutes for a quickfreeze of the pellet. The tube was then placed on freeze drierovernight. The dried particles were then resuspended in 1 mL of 70%ethanol and stored at 4° C. for future use.

Example 10: Administering Loaded YCWPs to Subject

The loaded YCWPs prepared according to Examples above are resuspended in1 mL of a solution suitable for injection, such as sterile water forinjection or sterile saline for injection, which optionally contains 5%human serum albumin, under sterile conditions. Once the loaded YCWPs arecarefully resuspended, the entire volume is drawn and injected to thedermis of a patient using a syringe.

Example 11: Preparing Dendritic Cells Containing Loaded Particles

The loaded YCWPs prepared according to Examples above in 70% ethanolsuspension are centrifuged. The ethanol is removed carefully andreplaced with 1 mL of PBS. The loaded YCWPs are sonicated. The loadedYCWPs are washed with sterile 1X PBS. After final wash, the loaded YCWPsare resuspended in PBS to approximately 1×10⁸ particles/100 μl PBS.

The loaded YCWPs are added to a dendritic cell culture at a ratio of1:100, and the culture was returned to 37° C. incubator. Subsequently,the following factors are added to the culture: 50 μg/mL of TNF-α insterile water is added to the culture at a ratio of 1:5000 in volume (2μL per 10 mL of culture); 10 μg/mL of IL-1β in sterile water is added tothe culture at a ratio of 1:1000 in volume; 10 μg/mL of IL-6 in sterilewater is added to the culture at a ratio of 1:1000 in volume; and 1mg/mL of PGE2 in 100% ethanol is added to the culture at a ratio of1:1000 in volume. After all factors are added and mixed into theculture, the culture is incubated overnight.

Example 12: Harvest of Dendritic Cells, Preparation and Cryopreservationof Vaccine Composition

The dendritic cell culture prepared according to Example 11 was removedfrom the incubator. The following procedure was performed in a hoodunder sterile conditions. 10 mL of media were removed from cultureflask. The culture flask was rinsed with 4.0-4.5 mL of 1×PBS and alsoadded to the media.

1.5-2.0 mL of CellStripper™ was added to the culture flask. The cultureflask was placed in 37° C. incubator for 10-20 minutes. About 4 mL ofthe culture media were added back to the flask from the tube to wash andremove cells. The flask was washed to harvest as many cells as possible.The cells were counted on hemacytometer or Cellometer™. The supernatantwas removed after centrifugation.

Subsequently, the cells were resuspended in CryoStor™ 10 at 5×10⁶cells/mL, aliquoted into cryovials properly labeled with patient IDNumber, date and cell concentration at 1.25×10⁶ cells/mL per vial (about250 μL). A 250-500 μL portion was saved in a cryovial for sterilitytesting, and the remaining vials were stored in Styrofoam containers andplaced under −86° C. to step down freeze.

Example 13: Preparation of the Solid Dose of Vaccine for PatientAdministration

One cryovial of patient's cell was removed from cryostorage andcarefully thawed at 37° C. in a water bath. Under sterile conditions, 1mL of sterile saline for injection with 5% human serum albumin (or 1 mLof sterile 1X PBS) was gently added to the cryovial containing thecells. After the cells were carefully resuspended, the entire volumefrom the cryovial was drawn and the syringe containing the vaccine wasused for administration to a patient.

Example 14: Immunization Procedure

To vaccinate a subject, a dose of 1.25 million dendritic cellscontaining vaccine loaded particles is cryopreserved in 0.2 mL of aserum-free, 10% dimethyl sulfoxide freezing medium (CryoStor™ CS-10,BioLife Solutinos, Inc.). Before injection, the dendritic cells isthawed and diluted to a 1 mL with sterile saline for injectioncontaining 5% human serum albumin (Albuminar −25, Aventis Behring). Thedilution is then transferred to a 3.0 cc syringe for injection and usinga needle no smaller than 23 gauge, which is administered within 2 hoursof the thawing. The injection can be administered subcutaneously into anarea of lymph nodes or administered intradermally.

Example 15: Isolation of Mononuclear Cells from Whole Peripheral BloodUsing the SepMate-50 System

Sepmate-50 tubes with inserts allow for quickly layering diluted bloodover the density gradient medium (LSM), and prevents the layers frommixing. After centrifugation with the brake on, enriched peripheralblood mononuclear cells (PBMC's) are poured into a fresh tube andprocessed as described below for future culturing

Procedure 1:

STEP PROCEDURE/WORK INSTRUCTIONS 1 Add 15 mL of lymphocyte separationmedium (LSM) to each SepMate tube by carefully, yet quickly, pipettingit through the central hole of each tube insert. 2 Pool the whole blood.3 Dilute the whole blood sample with twice the initial blood volume of1xPBS. 4 Add 30 mL diluted blood to the Sepmate tubes. 5 Centrifuge for10-15 minutes at room temperature. 6 Pour off the top layer containingthe enriched PBMC's from each Sepmate tube into new centrifuge tubes. 7Cap tubes and centrifuge 5 minutes. 8 Resuspend pellet in each tube withup to 1.0 mL ACK lysing buffer with pipette. Repeat cycles of adding 1XPBS to resuspend pellet and centrifuge. 9 Decant the supernatant, andaliquot 50 × 10⁶ cells suspended in 15 mL RPMI media per flask. Place inthe CO₂ incubator at 37° C. for 1-1.5 hours. 10 Remove flasks fromincubator. Wash pellet with 1X PBS. 11 Add 15 mL complete DC Media(containing IL-4, GM-CSF, and Gentamycin) and place in the 37° CO₂incubator for 22-24 hours. 12 On Day 2, continue with inoculation ofcultures with YWCP's and cytokines after the dendritic cells haveincubated at 37° C. for approx. 22 hours.

Procedure 2:

STEP PROCEDURE/WORK INSTRUCTIONS 1 Add 15 mL of lymphocyte separationmedium (LSM) to each SepMate-50 tube by carefully, yet quickly,pipetting it through the central hole of each tube insert. 2 Pool thewhole blood into a sterile 500 mL bottle or sterile 250 mL conicaltubes. 3 Dilute the whole blood sample with twice the initial bloodvolume of 1xPBS in the sterile 500 mL flask, and mix gently. 4 Add 30 mLdiluted blood by pipetting it smoothly and quickly down the side of theSepmate-50 tubes. 5 Centrifuge at 1200 g for 10-15 minutes at roomtemperature with the brake on. 6 Pour off the top layer containing theenriched PBMC's from each Sepmate tube into new sterile 50 mL centrifugetubes. 7 Cap tubes and centrifuge at 500 g for 5 minutes with brake on.8 Decant supernatant into waste bottle and resuspend pellet in each tubewith up to 1.0 mL ACK lysing buffer with pipette. 9 Combine into twosterile 50 mL tubes containing 5 mL of cell suspension each, and add5-10 mL 1xPBS to each tube. 10 Centrifuge again at 500 g for 5 minuteswith brake on. Decant the supernatant and resuspend the pellets withpipette in 10 mL 1xPBS. 11 Bring total volume in the tube(s) to 50 mLwith 1xPBS 12 Centrifuge at 200 g for 10 minutes with brake on. 13Decant supernatant into waste bottle, and resuspend pellet in 1xPBS upto the 50 mL demarcation on the tube, cap and mix well. 14 Remove anamount of the well-mixed suspension sufficient to perform a cell count,and record. 15 Centrifuge again at 200 g for 10 minutes with brake on.16 Decant the supernatant, and aliquot 50 × 10⁶ cells suspended in 15 mLRPMI media per flask. 17 Label the cell culture flasks with the patientID, date, and initials and place in the CO₂ incubator at 37° C. for1-1.5 hours. 18 Remove flasks from incubator and return them to thebiological safety hood. 19 Without touching the bottom of culture flask(do not disturb the adherent cells on the bottom), pipette off the RPMIand discard into waste container. 20 Carefully add 10 mL of 1xPBS downthe inner side of each culture flask and rock gently. 21 Pipette off thePBS containing any undesired non-adherent cells and discard into wastecontainer. 22 Repeat steps 20 and 21 two more times. 23 Gently add 15 mLcomplete DC Media (containing IL-4, GM- CSF, and Gentamycin) to eachflask by pipetting down the inner side of it. 24 Write “Culture Day 1”on the flasks, and place in the 37° CO₂ incubator for 22-24 hours. 25 OnDay 2, continue with inoculation of cultures with YWCP's and cytokines.

Example 16: Generation of Dendritic Cells Combined with Loaded YCWP's

Following the procedures in Example 15, the following methods areperformed:

-   -   I. Addition of YCWP

STEP PROCEDURE/WORK INSTRUCTIONS 1 To each dendritic cell culture flask,add a sufficient volume of loaded YCWP at a ratio of 1:100. (50-200 ul)and incubate for 1-2 hours.

-   -   II. Preparation And Addition of Cytokines

STEP PROCEDURE/WORK INSTRUCTIONS 1 Add TNF-α, 1β, IL-6 and PGE2 to eachculture flask,

Example 17: Harvest of Cells, Preparation and Cryopreservation ofVaccine Composition

The following methods are performed:

-   -   Harvest of cells:

STEP PROCEDURE/WORK INSTRUCTIONS 1 Add 4.0-4.5 mL 1xPBS to each flaskcontaining 10 mL media. 2 Add 1.5-2.0 mL CellStripper ™ to each flask torelease mature cells and incubate at 37° C. 3 Centrifuge and removesupernatant by decanting until “dry” pellet remains.

-   -   II. Preparation of Vaccine Composition and Cryopreservation:

STEP PROCEDURE/WORK INSTRUCTIONS 1 Resuspend cells in CryoStor ™ 10,aliquot and step down freeze.

Example 18: Experiment in Mouse Model

B16 murine tumor lysate loaded yeast cell wall particles was used as avaccine for mice. Mice not vaccinated and inoculated with one millionB16 tumor cells IV were used as control. Three days prior to tumorchallenge with the same tumor load IV, mice were vaccinated: (i) withtumor lysate and yeast cell wall particles simply mixed; or (ii) withyeast cell wall particles loaded with the tumor lysate. The totalprotein content of tumor lysate and the number of yeast cell walls forboth groups of the vaccinated mice were identical. 21 days followinginoculation with one million B16 tumor cells IV, the lungs of the miceof each group were examined.

FIGS. 6A, 6B and 6C show the results of the lungs of the mice of eachgroup 21 days following tumor challenge.

Example 19: Preparing Silicate Capped Yeast Cell Wall Particles

Yeast cell wall particles (YCWPs) were prepared and loaded with apeptide as described in the examples above. 1 mg of YCWPs were loadedwith 500 μg of the peptide. Subsequently, the freeze dried, loaded YCWPswere suspended in 1 ml of absolute ethanol, to which suspension 100 μlof tetraethylorthosilicate and 100 μl of a 10% aqueous ammonia solutionwere added. The mixture was shaken gently for 15 minutes at roomtemperature. The YCWPs were then washed thoroughly with absolute ethanoland kept in ethanol at 4° C. until use.

Example 20: In Vitro Leaking Assay

The YCWPs were loaded with fluorescence labeled albumin and then aportion of the loaded YCWPs were capped with silicate according to theexample above while others remain uncapped.

Both the uncapped and silicate capped YCWPs loaded with fluorescencelabeled albumin were first read on a reader to obtain the initialreading to the total fluorescence counts, and then both the uncapped andsilicate capped YCWPs were shaken vigorously. Supernatants were takenfrom both the uncapped and the silicate capped YCWPs at one hour and attwo hours to obtain the fluorescence readings, detailed below.

Silicate Capped PBS (control) Uncapped YCWPs YCWPs Initial fluorescence8879 59645 175861 counts Fluorescence counts 6329 14751 27797 ofsupernatants after 1-hour shaking Fluorescence counts 5944 9893 11700 ofsupernatants after 1-hour shaking

As shown in the table above, after one hour of shaking, uncapped YCWPsleaked 24.73% of the total fluorescence and silicate capped YCWPs leaked15.81% of the total fluorescence. After two hours of shaking, uncappedYCWPs leaked 16.6% of the total fluorescence and silicate capped YCWPsleaked 6.65% of the total fluorescence. In summary after two hours, theuncapped YCWPs lost 41.33% of the fluorescent labeled albumin while thesilicate capped YCWPs lost only 22.46% of the loaded albumin.

Example 21: In Vivo Loading Release Assay

Mouse macrophage Raw cells, a phagocytic monocytic cell line, wereplated in 6-well plate and cultured overnight. Uncapped YCWPs andsilicate capped YCWPs, both loaded with fluorescence labeled albumin,were added into the cells on the second day morning. After overnightculture, cells were washed several times with PBS and lysed. Lysateswere centrifuged to collect supernatants to obtain fluorescencereadings. The supernatant and pellet of the uncapped YCWPs hadfluorescence counts of 1042 and 1094 respectively; whereas thesupernatant and the pellet of the silicate capped YCWPs had fluorescencecounts of 1945 and 878 respectively. As such, the silicate capped YCWPsdelivered 86.6% more released albumin into the cytoplasm of thephagocytic cells than the uncapped YCWPs did.

Example 22: In Vitro Phagocytosis Assay

Mouse macrophage Raw cells were plated in 24-well plate and culturedovernight. Uncapped YCWPs and silicate capped YCWPs, both loaded withfluorescence labeled albumin were added into the cells on the second daymorning. Cell fluorescence readings were measured at 20 minutes, 1 hour,and 2 hours, respectively.

Fluorescence Counts 20 minutes 1 hour 2 hours Uncapped 10 μl 3560 31233193 YCWPs 30 μl 3485 3137 3571 60 μl 3579 3442 3928 Silicate capped 10μl 3499 3754 4624 YCWPs 30 μl 3408 4599 6213 60 μl 4408 6944 11065

As shown in the table above, the silicate capped YCWPs delivered 82%more loaded albumin into the phagocytic cells than the uncapped YCWPsdid.

Example 23: Mouse Survival Study

Survival study was performed on five groups of mice, with 5 mice in eachgroup of Groups I-IV and 10 mice in Group V. The control group (Group I)received 0.5×10⁶ B16 melanoma tumor cells by IV injection. The “regularYCWP” group (Group II) received 0.5×10⁶ B16 melanoma tumor cells by IVinjection and vaccination by interdermal injection one week prior to andeach week following tumor cell IV injection with uncapped YCWPs loadedwith B16 tumor lysate until week 6. The “Si capped YCWPs” group (GroupIII) received 0.5×10⁶ B16 melanoma tumor cells by IV injection andvaccination by interdermal injection one week prior to and each weekfollowing tumor cell IV injection with silicate capped YCWPs loaded withB16 tumor lysate until week 6. The “regular YCWPs+AD” group (Group IV)received 0.5×10⁶ B16 melanoma tumor cells by IV injection andvaccination by interdermal injection one week prior to and each weekfollowing tumor cell IV injection with uncapped YCWPs loaded with B16tumor lysate, and GpC Oligonucleotide and Monophosphoryl lipid Aadjuvants until week 6. The “Si capped YCWP+AD group” (Group V) received0.5×10⁶ B16 melanoma tumor cells by IV injection and vaccination byinterdermal injection one week prior to and each week following tumorcell IV injection with silicate capped YCWPs loaded with B16 tumorlysate, and GpC oligonucleotide and monophosphoryl lipid A adjuvantsuntil week 6.

As shown in FIG. 8, all mice in the control group died in about 22 days.All mice in the regular YCWP group died in about 25 days, all mice inthe silicate capped YCWP group died in about 35 days, and all mice inthe regular YCWP and adjuvant group dies in about 45 days. In contrast,about 40% of the mice in the silicate capped YCWP and adjuvant groupsurvived after 100 days.

1-10. (canceled)
 11. A method for producing an incubated dendritic cellcontaining a vaccine loaded particle, comprising: loading a biologicalmaterial into the particle to produce a biological material loadedparticle; (ii) freeze-drying the biological material loaded particle;and (iii) incubating the biological material loaded particle with adendritic cell, wherein the biological material comprises a protein or afragment thereof, nucleic acid, carbohydrate, tumor lysate, or acombination thereof; and wherein incubating the biological materialloaded particle with the dendritic cell causes the dendritic cell tophagocytose the biological material loaded particle.
 12. The method ofclaim 11, further comprising (a) resuspending the biological materialloaded particle in solution and (b) freeze-drying the resuspendedsolution before step (iii).
 13. The method of claim 11, wherein step(iii) comprises: (a) contacting the biological material loaded particlewith the dendritic cell at a ratio of from about 1:1 to about 100:1, (b)incubating the biological material loaded particle with the dendriticcell; and (c) collecting the dendritic cell containing the loadedparticle and washing the cell.
 14. The method of claim 13, wherein thebiological material loaded particle is incubated with the dendritic cellfor less than one hour. 15-31. (canceled)
 32. A method for producing acell mixture containing a yeast cell wall particle loaded with abiological material and capped with a silicate, comprising: (i) loadinga biological material into a yeast cell wall particle to produce aloaded particle wherein the biological material comprises a protein or afragment thereof, nucleic acid, carbohydrate, antigen or a combinationthereof, and wherein the biological material is not a tumor lysate,viral antigen, or bacterial antigen; (ii) capping the loaded particlewith a silicate; (iii) freeze-drying the capped, loaded particle; and(iv) incubating the capped, loaded particle with a cell of monocyticorigin wherein the incubation causes the cell of monocytic origin tophagocytose the capped, loaded particle.
 33. The method of claim 32,wherein the cell of monocytic origin is a pre-dendritic cell, adendritic cell, or a partially differentiated dendritic cell. 34.(canceled)
 35. The method of claim 32, wherein step (i) comprises: (a)suspending a yeast cell wall particle and a biological material in adiluent and incubating for a period of time to allow the biologicalmaterial to be absorbed by the yeast cell wall particle; and (b)freeze-drying the suspension to load the biological material within theyeast cell wall particle.
 36. The method of claim 35, wherein steps (a)and (b) are repeated at least once.
 37. (canceled)
 38. The method ofclaim 32, wherein the silicate is tetraethylorthosilicate,tetramethylorthosilicate, tetrapropylorthosilicate, ortetrabutylorthosilicate. 39-44. (canceled)
 45. A vaccine comprising (i)a yeast cell wall particle (YCWP), (ii) a viral antigen or bacterialantigen loaded into the yeast cell wall particle, and (ii) a silicate,wherein the YCWP is modified by capping with the silicate, wherein theviral antigen or bacterial antigen is not a tumor lysate, wherein theyeast cell wall particle is a hollow, porous structure, and wherein thevaccine does not comprise a cell of monocytic origin.
 46. The vaccine ofclaim 45, wherein the silicate is tetraethylorthosilicate,tetramethylorthosilicate, tetrapropylorthosilicate, ortetrabutylorthosilicate.
 47. The vaccine of claim 45, wherein thesilicate comprises an organic moiety attached to each of the four oxygencompounds of an orthosilicate.
 48. A method of treating an infectiousdisease, comprising administering to a subject the vaccine of claim 45.49. A method for treating cancer, comprising administering to a subjectthe vaccine of claim 45.